pflmi  (New England Biolabs)


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    PflMI
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    PflMI 5 000 units
    Catalog Number:
    R0509L
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    Category:
    Restriction Enzymes
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    5 000 units
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    New England Biolabs pflmi
    PflMI
    PflMI 5 000 units
    https://www.bioz.com/result/pflmi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pflmi - by Bioz Stars, 2021-04
    93/100 stars

    Images

    1) Product Images from "Comparison of TALEN scaffolds in Xenopus tropicalis"

    Article Title: Comparison of TALEN scaffolds in Xenopus tropicalis

    Journal: Biology Open

    doi: 10.1242/bio.20136676

    Disruption of restriction enzyme recognition sites between the left and right target sites in embryos injected with TALEN mRNAs. (A,B) Schematic drawing of the genomic PCR product containing Tyr I (A) or Tyr II (B) target sites. An HaeIII site (Hae) and a PflMI site (Pfl) are located in the spacer sequences of Tyr I and Tyr II, respectively. The primer sets and target sites are indicated as blue arrowheads and red bars, respectively. (C–F) Quantification of resistance to restriction enzyme digestion. Embryos were injected with 400 pg, 80 pg or 0 pg (control) of TALEN-Tyr I mRNAs (C,D) or TALEN-Tyr II mRNAs (E,F). Genomic DNA was separately prepared from randomly selected embryos at NF-stage 35/36 ( Fig. 2 ; Fig. 3 ) and subjected to PCR using a specific primer set to amplify DNA fragments containing the target sites. The PCR products were digested with HaeIII (C,D) or PflMI (E,F) enzymes and separated on agarose gels. The injected TALEN scaffolds are shown at the bottom. The number of analyzed embryos is shown at the top of each column. Due to embryo death, genomic DNA was not extracted from embryos injected with 400 pg of ΔNΔC-Tyr II mRNAs (E). The statistical significance compared to the control (a) or embryos injected with ΔNΔC-ELD/KKR mRNAs (b) was assessed using a Tukey test. P
    Figure Legend Snippet: Disruption of restriction enzyme recognition sites between the left and right target sites in embryos injected with TALEN mRNAs. (A,B) Schematic drawing of the genomic PCR product containing Tyr I (A) or Tyr II (B) target sites. An HaeIII site (Hae) and a PflMI site (Pfl) are located in the spacer sequences of Tyr I and Tyr II, respectively. The primer sets and target sites are indicated as blue arrowheads and red bars, respectively. (C–F) Quantification of resistance to restriction enzyme digestion. Embryos were injected with 400 pg, 80 pg or 0 pg (control) of TALEN-Tyr I mRNAs (C,D) or TALEN-Tyr II mRNAs (E,F). Genomic DNA was separately prepared from randomly selected embryos at NF-stage 35/36 ( Fig. 2 ; Fig. 3 ) and subjected to PCR using a specific primer set to amplify DNA fragments containing the target sites. The PCR products were digested with HaeIII (C,D) or PflMI (E,F) enzymes and separated on agarose gels. The injected TALEN scaffolds are shown at the bottom. The number of analyzed embryos is shown at the top of each column. Due to embryo death, genomic DNA was not extracted from embryos injected with 400 pg of ΔNΔC-Tyr II mRNAs (E). The statistical significance compared to the control (a) or embryos injected with ΔNΔC-ELD/KKR mRNAs (b) was assessed using a Tukey test. P

    Techniques Used: Injection, Polymerase Chain Reaction

    Target site sequences in TALEN-mRNA-injected embryos. Target DNA sequences were determined using pooled genomic DNAs purified from NF-stage 35/36 embryos that had been injected with 400 pg of TALEN-Tyr I (A) or –Tyr II (B) mRNAs. (A) Mutation of target site sequences by TALEN-Tyr I. Genomic DNA was extracted from each of 16, 9, 13 and 13 embryos injected with TAL-Tyr I, ΔNΔC-Tyr I, ΔNΔC-ELD/KKR-Tyr I and ΔNΔC-ELD-S/KKR-S-Tyr I mRNAs, respectively, and pooled. (B) Mutation of target site sequences by TALEN-Tyr II. Genomic DNA was extracted from each of 7, 12 and 9 embryos injected with TAL-Tyr II, ΔNΔC-ELD/KKR-Tyr II and ΔNΔC-ELD-S/KKR-S-Tyr II mRNAs, respectively, and pooled. Due to embryo death, genomic DNA was not purified from embryos injected with ΔNΔC-Tyr II mRNAs. The wild-type sequence is shown as WT. The black bars indicate the Tyr I (A) and Tyr II (B) target sites. Gaps resulting from deletion are denoted as dashes. Inserted nucleotides are indicated as red characters. The HaeIII (A) and PflMI (B) recognition sequences are indicated as blue characters. The mutation types and frequencies are indicated on the right. (C,D) Percentage of mutant target site sequences derived from embryos injected with TALEN-Tyr I (C) or TALEN-Tyr II (D) mRNAs.
    Figure Legend Snippet: Target site sequences in TALEN-mRNA-injected embryos. Target DNA sequences were determined using pooled genomic DNAs purified from NF-stage 35/36 embryos that had been injected with 400 pg of TALEN-Tyr I (A) or –Tyr II (B) mRNAs. (A) Mutation of target site sequences by TALEN-Tyr I. Genomic DNA was extracted from each of 16, 9, 13 and 13 embryos injected with TAL-Tyr I, ΔNΔC-Tyr I, ΔNΔC-ELD/KKR-Tyr I and ΔNΔC-ELD-S/KKR-S-Tyr I mRNAs, respectively, and pooled. (B) Mutation of target site sequences by TALEN-Tyr II. Genomic DNA was extracted from each of 7, 12 and 9 embryos injected with TAL-Tyr II, ΔNΔC-ELD/KKR-Tyr II and ΔNΔC-ELD-S/KKR-S-Tyr II mRNAs, respectively, and pooled. Due to embryo death, genomic DNA was not purified from embryos injected with ΔNΔC-Tyr II mRNAs. The wild-type sequence is shown as WT. The black bars indicate the Tyr I (A) and Tyr II (B) target sites. Gaps resulting from deletion are denoted as dashes. Inserted nucleotides are indicated as red characters. The HaeIII (A) and PflMI (B) recognition sequences are indicated as blue characters. The mutation types and frequencies are indicated on the right. (C,D) Percentage of mutant target site sequences derived from embryos injected with TALEN-Tyr I (C) or TALEN-Tyr II (D) mRNAs.

    Techniques Used: Injection, Purification, Mutagenesis, Sequencing, Derivative Assay

    2) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.
    Figure Legend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Techniques Used: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Comparison of TALEN scaffolds in Xenopus tropicalis
    Article Snippet: The second PCR was performed using Tyr IF and Tyr IR for Tyr I or Tyr IIF and Tyr IIR for Tyr II , with 20 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 min. .. The PCR products were digested with HaeIII (TOYOBO) or PflMI (NEB) to examine the efficiency of targeted gene disruption in the injected embryos. .. The products were analyzed by gel electrophoresis, and the brightness of each band was measured; the molecular ratio was calculated as the quotient of the brightness divided by the nucleotide length.

    Article Title: Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity
    Article Snippet: Heavy chain PCR products were extracted from 2% agarose gels and inserted into the pCR4-TOPO vector (Invitrogen). .. Light chain PCR products were purified with QIAquick and digested with restriction enzymes PflFI or PflmI (New England Biolabs, Inc.) to disrupt the rearranged Vk 21-12 gene transcribed by the SP2/0 fusion partner (only three other kappa genes have both restriction sites). .. After 2% agarose gel electrophoresis of the PCR products, the uncut bands were extracted and inserted into pCR-4-TOPO for sequencing.

    Injection:

    Article Title: Comparison of TALEN scaffolds in Xenopus tropicalis
    Article Snippet: The second PCR was performed using Tyr IF and Tyr IR for Tyr I or Tyr IIF and Tyr IIR for Tyr II , with 20 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 min. .. The PCR products were digested with HaeIII (TOYOBO) or PflMI (NEB) to examine the efficiency of targeted gene disruption in the injected embryos. .. The products were analyzed by gel electrophoresis, and the brightness of each band was measured; the molecular ratio was calculated as the quotient of the brightness divided by the nucleotide length.

    Purification:

    Article Title: Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity
    Article Snippet: Heavy chain PCR products were extracted from 2% agarose gels and inserted into the pCR4-TOPO vector (Invitrogen). .. Light chain PCR products were purified with QIAquick and digested with restriction enzymes PflFI or PflmI (New England Biolabs, Inc.) to disrupt the rearranged Vk 21-12 gene transcribed by the SP2/0 fusion partner (only three other kappa genes have both restriction sites). .. After 2% agarose gel electrophoresis of the PCR products, the uncut bands were extracted and inserted into pCR-4-TOPO for sequencing.

    other:

    Article Title: Cdk1 uncouples CtIP-dependent resection and Rad51 filament formation during M-phase double-strand break repair
    Article Snippet: PflMI and terminal transferase were obtained from New England Biolabs, Inc. Histone H1 (382150) was obtained from EMD.

    Plasmid Preparation:

    Article Title: Lack of Functional Receptors Is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells
    Article Snippet: We generated a full-length infectious provirus molecular clone of CAEV by using the recently described recombinant plasmids pK9Kb and pBSΔ ( ). .. Double digestion of pK9Kb plasmid DNA with Sal I (Promega, Charbonnieres-Les-Bains, France) and Pflm I (New England Biolabs, Saint-Quentin Yvelines, France) endonucleases under the conditions recommended by the supplier released an 8.4-kb fragment corresponding to the complete CAEV-CO genome lacking the 3′ end of the env gene and the 3′ long terminal repeat (LTR). .. This fragment was separated by gel electrophoresis and harvested by the freeze-and-squeeze method.

    Activity Assay:

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles
    Article Snippet: XI sites, allowing for directional assembly into a full-length replicon cDNA by in vitro ligation ( Serial deletions within the TGEV structural gene region were generated from the unique Pfl MI site at the very 3′ end of the GFP gene and extended for various distances toward the 3′ end of the genome (Fig. ). .. TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase. ..

    Generated:

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles
    Article Snippet: XI sites, allowing for directional assembly into a full-length replicon cDNA by in vitro ligation ( Serial deletions within the TGEV structural gene region were generated from the unique Pfl MI site at the very 3′ end of the GFP gene and extended for various distances toward the 3′ end of the genome (Fig. ). .. TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase. ..

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    New England Biolabs pflmi
    Disruption of restriction enzyme recognition sites between the left and right target sites in embryos injected with TALEN mRNAs. (A,B) Schematic drawing of the genomic <t>PCR</t> product containing Tyr I (A) or Tyr II (B) target sites. An HaeIII site (Hae) and a <t>PflMI</t> site (Pfl) are located in the spacer sequences of Tyr I and Tyr II, respectively. The primer sets and target sites are indicated as blue arrowheads and red bars, respectively. (C–F) Quantification of resistance to restriction enzyme digestion. Embryos were injected with 400 pg, 80 pg or 0 pg (control) of TALEN-Tyr I mRNAs (C,D) or TALEN-Tyr II mRNAs (E,F). Genomic DNA was separately prepared from randomly selected embryos at NF-stage 35/36 ( Fig. 2 ; Fig. 3 ) and subjected to PCR using a specific primer set to amplify DNA fragments containing the target sites. The PCR products were digested with HaeIII (C,D) or PflMI (E,F) enzymes and separated on agarose gels. The injected TALEN scaffolds are shown at the bottom. The number of analyzed embryos is shown at the top of each column. Due to embryo death, genomic DNA was not extracted from embryos injected with 400 pg of ΔNΔC-Tyr II mRNAs (E). The statistical significance compared to the control (a) or embryos injected with ΔNΔC-ELD/KKR mRNAs (b) was assessed using a Tukey test. P
    Pflmi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pflmi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pflmi - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

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    Disruption of restriction enzyme recognition sites between the left and right target sites in embryos injected with TALEN mRNAs. (A,B) Schematic drawing of the genomic PCR product containing Tyr I (A) or Tyr II (B) target sites. An HaeIII site (Hae) and a PflMI site (Pfl) are located in the spacer sequences of Tyr I and Tyr II, respectively. The primer sets and target sites are indicated as blue arrowheads and red bars, respectively. (C–F) Quantification of resistance to restriction enzyme digestion. Embryos were injected with 400 pg, 80 pg or 0 pg (control) of TALEN-Tyr I mRNAs (C,D) or TALEN-Tyr II mRNAs (E,F). Genomic DNA was separately prepared from randomly selected embryos at NF-stage 35/36 ( Fig. 2 ; Fig. 3 ) and subjected to PCR using a specific primer set to amplify DNA fragments containing the target sites. The PCR products were digested with HaeIII (C,D) or PflMI (E,F) enzymes and separated on agarose gels. The injected TALEN scaffolds are shown at the bottom. The number of analyzed embryos is shown at the top of each column. Due to embryo death, genomic DNA was not extracted from embryos injected with 400 pg of ΔNΔC-Tyr II mRNAs (E). The statistical significance compared to the control (a) or embryos injected with ΔNΔC-ELD/KKR mRNAs (b) was assessed using a Tukey test. P

    Journal: Biology Open

    Article Title: Comparison of TALEN scaffolds in Xenopus tropicalis

    doi: 10.1242/bio.20136676

    Figure Lengend Snippet: Disruption of restriction enzyme recognition sites between the left and right target sites in embryos injected with TALEN mRNAs. (A,B) Schematic drawing of the genomic PCR product containing Tyr I (A) or Tyr II (B) target sites. An HaeIII site (Hae) and a PflMI site (Pfl) are located in the spacer sequences of Tyr I and Tyr II, respectively. The primer sets and target sites are indicated as blue arrowheads and red bars, respectively. (C–F) Quantification of resistance to restriction enzyme digestion. Embryos were injected with 400 pg, 80 pg or 0 pg (control) of TALEN-Tyr I mRNAs (C,D) or TALEN-Tyr II mRNAs (E,F). Genomic DNA was separately prepared from randomly selected embryos at NF-stage 35/36 ( Fig. 2 ; Fig. 3 ) and subjected to PCR using a specific primer set to amplify DNA fragments containing the target sites. The PCR products were digested with HaeIII (C,D) or PflMI (E,F) enzymes and separated on agarose gels. The injected TALEN scaffolds are shown at the bottom. The number of analyzed embryos is shown at the top of each column. Due to embryo death, genomic DNA was not extracted from embryos injected with 400 pg of ΔNΔC-Tyr II mRNAs (E). The statistical significance compared to the control (a) or embryos injected with ΔNΔC-ELD/KKR mRNAs (b) was assessed using a Tukey test. P

    Article Snippet: The PCR products were digested with HaeIII (TOYOBO) or PflMI (NEB) to examine the efficiency of targeted gene disruption in the injected embryos.

    Techniques: Injection, Polymerase Chain Reaction

    Target site sequences in TALEN-mRNA-injected embryos. Target DNA sequences were determined using pooled genomic DNAs purified from NF-stage 35/36 embryos that had been injected with 400 pg of TALEN-Tyr I (A) or –Tyr II (B) mRNAs. (A) Mutation of target site sequences by TALEN-Tyr I. Genomic DNA was extracted from each of 16, 9, 13 and 13 embryos injected with TAL-Tyr I, ΔNΔC-Tyr I, ΔNΔC-ELD/KKR-Tyr I and ΔNΔC-ELD-S/KKR-S-Tyr I mRNAs, respectively, and pooled. (B) Mutation of target site sequences by TALEN-Tyr II. Genomic DNA was extracted from each of 7, 12 and 9 embryos injected with TAL-Tyr II, ΔNΔC-ELD/KKR-Tyr II and ΔNΔC-ELD-S/KKR-S-Tyr II mRNAs, respectively, and pooled. Due to embryo death, genomic DNA was not purified from embryos injected with ΔNΔC-Tyr II mRNAs. The wild-type sequence is shown as WT. The black bars indicate the Tyr I (A) and Tyr II (B) target sites. Gaps resulting from deletion are denoted as dashes. Inserted nucleotides are indicated as red characters. The HaeIII (A) and PflMI (B) recognition sequences are indicated as blue characters. The mutation types and frequencies are indicated on the right. (C,D) Percentage of mutant target site sequences derived from embryos injected with TALEN-Tyr I (C) or TALEN-Tyr II (D) mRNAs.

    Journal: Biology Open

    Article Title: Comparison of TALEN scaffolds in Xenopus tropicalis

    doi: 10.1242/bio.20136676

    Figure Lengend Snippet: Target site sequences in TALEN-mRNA-injected embryos. Target DNA sequences were determined using pooled genomic DNAs purified from NF-stage 35/36 embryos that had been injected with 400 pg of TALEN-Tyr I (A) or –Tyr II (B) mRNAs. (A) Mutation of target site sequences by TALEN-Tyr I. Genomic DNA was extracted from each of 16, 9, 13 and 13 embryos injected with TAL-Tyr I, ΔNΔC-Tyr I, ΔNΔC-ELD/KKR-Tyr I and ΔNΔC-ELD-S/KKR-S-Tyr I mRNAs, respectively, and pooled. (B) Mutation of target site sequences by TALEN-Tyr II. Genomic DNA was extracted from each of 7, 12 and 9 embryos injected with TAL-Tyr II, ΔNΔC-ELD/KKR-Tyr II and ΔNΔC-ELD-S/KKR-S-Tyr II mRNAs, respectively, and pooled. Due to embryo death, genomic DNA was not purified from embryos injected with ΔNΔC-Tyr II mRNAs. The wild-type sequence is shown as WT. The black bars indicate the Tyr I (A) and Tyr II (B) target sites. Gaps resulting from deletion are denoted as dashes. Inserted nucleotides are indicated as red characters. The HaeIII (A) and PflMI (B) recognition sequences are indicated as blue characters. The mutation types and frequencies are indicated on the right. (C,D) Percentage of mutant target site sequences derived from embryos injected with TALEN-Tyr I (C) or TALEN-Tyr II (D) mRNAs.

    Article Snippet: The PCR products were digested with HaeIII (TOYOBO) or PflMI (NEB) to examine the efficiency of targeted gene disruption in the injected embryos.

    Techniques: Injection, Purification, Mutagenesis, Sequencing, Derivative Assay

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Journal: Nucleic Acids Research

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    doi: 10.1093/nar/gkl179

    Figure Lengend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Article Snippet: To create linear fragments of differing sizes containing a single Tnp recognition ES, pWSR6103 was digested with PflMI and either XmnI, AatII, NdeI, NarI, BglI or PvuII (all NEB).

    Techniques: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    Construction of pBSCA and pBSCAδE5 molecular clones. pK9Kb plasmid DNA was double digested with Sal I and Pflm I to release an 8.4-kb fragment corresponding to the complete CAEV-CO genome lacking the 3′ end of the env gene and 3′ LTR. This fragment was gel purified and then inserted into the pBSΔ plasmid DNA doubly digested with Xho I and Pflm I. The isolated molecular clone corresponds to a reconstituted complete infectious proviral CAEV-CO genome (pBSCA). pBSCAδE5 was derived from the pBSCA plasmid by deletion of an 0.4-kb Bam HI fragment in the envelope-coding sequence.

    Journal: Journal of Virology

    Article Title: Lack of Functional Receptors Is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells

    doi:

    Figure Lengend Snippet: Construction of pBSCA and pBSCAδE5 molecular clones. pK9Kb plasmid DNA was double digested with Sal I and Pflm I to release an 8.4-kb fragment corresponding to the complete CAEV-CO genome lacking the 3′ end of the env gene and 3′ LTR. This fragment was gel purified and then inserted into the pBSΔ plasmid DNA doubly digested with Xho I and Pflm I. The isolated molecular clone corresponds to a reconstituted complete infectious proviral CAEV-CO genome (pBSCA). pBSCAδE5 was derived from the pBSCA plasmid by deletion of an 0.4-kb Bam HI fragment in the envelope-coding sequence.

    Article Snippet: Double digestion of pK9Kb plasmid DNA with Sal I (Promega, Charbonnieres-Les-Bains, France) and Pflm I (New England Biolabs, Saint-Quentin Yvelines, France) endonucleases under the conditions recommended by the supplier released an 8.4-kb fragment corresponding to the complete CAEV-CO genome lacking the 3′ end of the env gene and the 3′ long terminal repeat (LTR).

    Techniques: Clone Assay, Plasmid Preparation, Purification, Isolation, Derivative Assay, Sequencing

    Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Construct, Sequencing, Labeling, Recombinant, In Vitro

    Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Sequencing, Isolation, TA Cloning, Generated, Transfection, Derivative Assay, Infection, Ligation